Engineered antibodies based on murine monoclonal antibodies directed against carcinoembryonic antigen (CEA) will be generated for use in the diagnosis and therapy of colon carcinoma and other solid tumors in patients. the use of murine monoclonal antibodies has been limited by their immunogenicity and lack of effector functions. Many of these shortcomings have been overcome by the use of genetic engineering to produce chimeric mouse/human antibodies. We have produced chimeric antibodies based on T84.66, a high-affinity, highly specific anti-CEA antibody; these are available for therapy studies. During the upcoming project period we propose to develop additional engineered antibodies that differ in key properties such as size, valency, and affinity, such that the effects of these characteristics on biodistribution and therapeutic index can be evaluated. T84.12 is an additional anti-CEA antibody which demonstrates improved biodistribution compared to T84.66. cDNA clones encoding the heavy and light chains of T84.12 have been cloned and chimerized using polymerase chain reaction methods. Genetically engineered fragments (Fab, F(ab')2), single chain antibodies, CH2 deletion, and minibody will be developed during the next project period. Based on results on immune responses to radiolabeled antibodies in Project 0005, Human Studies, antibodies with reduced immunogenicity will be developed. Specific mutations will be introduced to permit site- specific chemical conjugation (ser to cys mutations) and glycosylation (asn-X-ser/thr), to facilitate coupling of chelate or protecting groups to modulate half-life and immunogenicity. Stably transfected cell lines producing engineered antibodies at high level will be generated and transferred to Core 9003. Bacterial expression will be used for single chain antibody and derivatives. These studies will contribute to the rational design of antibody/chelate/radiometal agents for use in radioimmunotherapy of colorectal cancer.